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Cationic Vectors in Ocular Drug Delivery

LAURA RABINOVICH-GUILATTa,b, PATRICK COUVREURa, GREGORY LAMBERTb and CATHERINE DUBERNET a,*

aUMR CNRS 8612, School of Pharmacy, Chatenay Malabry, 92296, France; bNovagali Pharma SA, Evry, 91058, France

(Received 13 July 2004; Revised 30 September 2004; Accepted 1 October 2004)

Despite extensive research in the field, the major problem in the ocular drug delivery domain still israpid precorneal drug loss and poor corneal permeability. One of the approaches recently developed isthe drug incorporation into cationic submicronic vectors which exploit the negative charges present atthe corneal surface for increased residence time and penetration.

This review will focus on the formulation of three main representative cationic colloids developed forophthalmic delivery: liposomes, emulsions and nanoparticles (NP). Parameters such as choice of thevector type and size, nature of the cationic molecule, pH and ionic strength of the external phase andcharacteristics of the encapsulated drug will be discussed with accent on the relevance of the positivecharge.

Keywords: Ocular drug delivery; Ophthalmic dosage forms; Nanoparticles; Emulsions; Liposomes;Cationic colloids

INTRODUCTION

Whenever an ophthalmic drug is applied topically to the

eye, only a small amount (,5%) actually penetrates the

cornea and reaches the internal anterior tissue of the eyes.

The amount of drug that ultimately penetrates the cornea

is often determined during the first 4–6 min after topical

dosing. Precorneal factors, such as rapid and efficient

drainage by the nasolacrimal apparatus and non-corneal

absorption, partially explain this phenomenon. In addition,

the relative impermeability of the cornea to both

hydrophilic and hydrophobic molecules accounts for the

poor ocular bioavailability and systemic adverse effects as

well. As a result, optimal absorption depends on achieving

a satisfactory and rapid penetration rate across the cornea

to minimize the competing, but non-absorptive factors.

Basic research concerning the physicochemical proper-

ties of the tears and cornea and their potential impact on

ocular delivery was performed in the 70’s and 80’s (Patton

and Robinson, 1976; Ahmed and Patton, 1984; Ahmed

et al., 1987; Rojanasakul and Robinson, 1989), and this

knowledge is still exploited now in the development of

new ophthalmic delivery systems. The various approaches

that have been attempted to increase the bioavailability

and the duration of the therapeutic action of ocular drugs

can be divided into two categories (Ding, 1998). The first

one is based on the use of sustained drug delivery systems,

which provide the controlled and continuous delivery of

ophthalmic drugs (implants, inserts, colloids). The second

involves maximizing corneal drug absorption and

minimizing precorneal drug loss (viscosity and pene-

tration enhancers, prodrugs, colloids). Cationic disper-

sions can provide simultaneously both advantages, by

interacting with the negatively charged corneal surface

components and the epithelium cellular membrane as will

be discussed in detail later. In addition, their adminis-

tration via conventional liquid dosage form is an attractive

feature for patient acceptability and compliance.

This paper will report the published studies concerning

cationic ocular delivery systems, especially those asses-

sing the relevance of the positive charge in the ocular

biodisposition and/or therapeutic efficacy. The aim of this

review is also to set up the relevant factors to take into

consideration when formulating such colloıdal delivery

systems, with special concern to the optimization of

physicochemical parameters related to the colloid charge.

ISSN 1061-186X print/ISSN 1029-2330 online q 2004 Taylor & Francis Ltd

DOI: 10.1080/10611860400015910

*Corresponding author. Address: Universite Paris Sud, Faculte de Pharmacie, 5 rue JB Clement, 92296 Chatenay Malabry, France.Tel.: þ33-1-46-83-53-86. Fax: þ33-1-46-61-93-34. E-mail: catherine.dubernet@cep.u-psud.fr

Journal of Drug Targeting, October–December 2004 Vol. 12 (9–10), pp. 623–633

OCULAR ABSORPTION: PHYSIOLOGY

AND MECHANISM

Corneal Structure

The cornea (500mm thick and 1.5 cm2 surface in the

normal adult) is anatomically composed of (from the

external to the internal side) the epithelium, Bowman’s

membrane, stroma, Descement’s membrane and endo-

thelium, but only the epithelium, stroma, and endothelium

represent barriers to drug absorption (Fig. 1). Usually, the

lipophilic cornea1 epithelium (6–7 cell layers and 50mm

thick) is the main barrier to drug absorption into the eye

with its tight junctions serving as a selective barrier for

small molecules and completely preventing the diffusion

of macromolecules via the paracellular pathway (Maren

and Jankowska, 1985). Moreover, as the rate of epithelial

turnover is approximately one cell layer per day

(Robinson, 1993), the outermost cells are highly

keratinized. The stroma (450mm) is a highly hydrophilic

tissue which consists mostly of water. Due to a relatively

open structure, drugs with molecular size up to 50,000 Da

can diffuse in normal stroma. Only for the most lipophilic

drugs, may the hydrophilic stroma represent the rate-

limiting barrier to ocular absorption (Huang et al., 1983;

Maren and Jankowska, 1985). The moderately lipophilic

cornea1 endothelium is a single layer of hexagonal cells

covering the posterior surface of the cornea, with less tight

junctions than at the epithelium.

For most ocular applied drugs, passive diffusion is

thought to be the main transport process across the cornea.

While lipophilic drugs prefer the transcellular pathway,

hydrophilic ones penetrate primarily through the para-

cellular pathway that involves passive or altered diffusion

through intercellular spaces.

The physicochemical properties of the drug (or of its

delivery system, when relevant), such as lipophilicity,

solubility, molecular size and shape, charge and degree of

ionization, affect the drug transport pathway and transport

rate across the tissue, as will be discussed later in this

review.

Corneal Charge

Most epithelia are known to possess permselective

properties, i.e. the ability to discriminate or to show

preference to the passage of charged molecules. In

addition to the intrinsic cellular membrane negative

charge, a layer of the glycoprotein mucin (a mixture of

neutral and acidic mucopolysaccharides) secreted by

goblet cells at the conjunctival surface is adjacent to the

corneal epithelium. Permselectivity is therefore a complex

phenomenon which combines not only a passive

contribution of membrane fixed charges such as ionizable

protein amino acid residues, stroma collagen and

proteoglycans, but also an active potential distribution

from cell membrane activity. As a consequence of the

above described facts, the cornea is negatively charged

and more permeable to cations than to anions at normal

pH, with an isoelectric point of 3.2 (Rojanasakul and

Robinson, 1989; Liaw et al., 1992). When an eyedrop is

formulated near neutral pH, cationic compounds thus

penetrate through the cornea easier than anionic species

(Liaw et al., 1992).

Tears Physiology

The normal precorneal tear film has a thickness of about

4–9mm and a volume of 7–10ml, although the eye can

retain up to 30ml without overflowing if care is taken not

to blink. The film is a trilaminar structure with each layer

distinctive in its own function. The anterior (outer) layer is

made up predominantly of lipids which reduce tears

evaporation, the middle layer (98% of the film) is

composed mainly of water, electrolytes and various

proteins and the posterior layer underlining the cornea is a

glycoprotein (mucin) layer which adds stability to the tear

film (Tiffany, 1994; Baeyens and Gurny, 1997).

FIGURE 1 Corneal surface and tear film structure. (1) Precorneal tears film (a: anterior lipid layer, b: aqueous layer, c: mucin coacervate, d: adsorbedmucin or mucous layer), (2) epithelial cell, (3) Bowman’s membrane, (4) stroma, (5) Descernet’s membrane and (6) endothelium. Scaled scheme,adapted from Robinson (1993).

L. RABINOVICH-GUILATT et al.624

Drainage of tears and instilled solutions away from the

front of the eye is an extremely efficient process (Fig. 2).

As an example, in normal awake rabbits, drainage

accounts for the loss of as much as 75% of the instilled

positive liposomal suspension within 1 min after instilla-

tion of a 25ml dose (Fitzgerald et al., 1987).

Tears Buffering Capacity

Instillation of 20ml of 0.067 M buffer pH 5.5 to human

volunteers was shown to result in an immediate tear pH

neutralization to pH 6.0–6.5, indicative of the limited but

still existing tears buffering capacity (Yamada et al.,

1998). Because of the small tear volume, topical

administration of any dose of a moderately strong buffer

is expected to momentarily overwhelm the ocular fluids

and to establish the pH in the eye. The variation on the tear

film pH following administration depends on both the

buffering capacity and the pH of the instilled solution (Hill

and Carney, 1980; Ahmed and Patton, 1984; Carney et al.,

1989). Subsequent rate of rise/drop in tear pH depends on

many aspects including factors affecting fluids dynamics

in the precorneal area (such as tears formation and

draining rates), formulation variables (irritation, pH and

ionic strength) and physical-chemical characteristics of

the tears (composition, buffering capacity).

Non-productive Absorption

Topically applied drugs that are neither lost through the

drainage apparatus nor absorbed by the cornea are

potentially available to be absorbed by the conjunctiva and

the sclera which show higher permeability than the cornea

(Chien et al., 1990; Ashton et al., 1991; Hamalainen et al.,

1997). These routes are commonly referred as non-

productive since the rapid local circulation rapidly

removes the compound from the area (Fig. 2); they are,

however, in some cases not totally unwanted as

therapeutic levels in posterior ocular tissues can be

achieved by this way (Ahmed and Patton, 1985; Chien

et al., 1990). Ahmed and Patton demonstrated for instance

that for timolol the aqueous humor level reflected the

corneal absorption while 70–80% of the iris-ciliary body

and vitreous humor concentrations indicated non corneal

routes (Ahmed and Patton, 1985). Patton and Robinson

have shown that the amount of drug absorbed by the

productive (corneal) route is insignificant when compared

with the overall loss of the drug (drainage þ non-

productive absorption), demonstrating that tear sampling

studies assessing drug disappearance evaluate mainly the

non-productive route (Patton and Robinson, 1976). It

should be noted that as the conjunctival absorption is less

influenced by the molecule size and lipophilicity than the

cornea, administration of lipophilic drugs not only

promotes the corneal permeability but also increases the

corneal to conjunctival relative selectivity (Chien et al.,

1990; Hamalainen et al., 1997). Such improved

distribution was also reported for colloidal carriers

(Calvo et al., 1994).

ADVANTAGES OF CATIONIC OVER NEUTRALOR ANIONIC DELIVERY SYSTEMS

A large variety of submicron-sized colloidal carriers have

been developed so far in the ophthalmic drug delivery field.

Figure 3 illustrates the most representative ones.

Nanoparticles (NP) are polymeric colloidal particles, the

biologically active molecule being encapsulated within

their polymeric matrix or simply adsorbed or conjugated

onto their surface. They can be further classified into

matricial type nanospheres (NS) or reservoir-type

nanocapsules (NC) filled with either oil or water core.

Liposomes are composed of single (SUV—small uni-

lamellar vesicles) or multiple (MLV—multi lamellar

vesicles) phospholipid bilayers surrounding water

compartments. Oil in water (o/w) submicron emulsions

are surfactant-stabilized oil droplets dispersed within an

external water phase.

FIGURE 2 Typical fate of a topically administered ophthalmicformulation.

FIGURE 3 Schematic representation of diverse types of cationic colloidal systems employed for drug delivery to ocular tissues.

CATIONIC VECTORS IN OCULAR DRUG DELIVERY 625

Studies Performed in Drug Ocular Delivery

Comparing Cationic and Anionic Vectors

Many studies performed in the last few years comparing

cationic to anionic ocular drug delivery systems have

confirmed the superiority of positively-charged colloids in

delivering therapeutic agents to the eye. It is noteworthy,

that while these experiments have confronted net positive

to either negative or neutral charge, none has evaluated the

effect of charge magnitude or density on the drug efficacy.

While data related to cationic liposomes were reviewed

extensively in previous references (Meisner and Mezer,

1995; Kaur et al., 2004), no comprehensive revision of other

colloidal systems has been done. Table I and the next

paragraphs summarize the most important and updated

findings concerning liposomes together with complete

results regarding charge effects in other ophthalmic colloids.

Liposomes

Schaeffer and Krohn have investigated the effect of

the charge of the colloid on the corneal uptake in vitro,

by tracing both 14C-liposomal phosphatidylcholine and14C-encapsulated penicillin (Schaeffer and Krohn, 1982).

They demonstrated that liposomes were taken up by the

cornea in the order of positive . negative . neutral and

that the most effective formulation (positive SUV)

produced a four-fold increase in the transcorneal flux of

penicillin G.

In another study, the in vivo aqueous humor

concentrations of liposomal acyclovir was significantly

improved by the cationic charge, even if preliminary

in vitro permeability studies had shown that the cornea

was less permeable to acyclovir in SA-liposomes

compared to negative ones or to acyclovir solution

(Law et al., 2000). This apparent discrepancy between

in vitro and in vivo results (lower in vitro permeation rate

together with an in vivo higher Cmax) suggested that the

absorption increase was rather due in vivo to a raised

residence time than to an augmented permeability.

Similarly, the bioavailability of tropicamide as assessed

by its pupil dilatatory effect in the rabbit eye showed that

the drug loaded on positively charged liposomes was more

TABLE I Published studies comparing pharmacokinetic and/or pharmacodynamic parameters of negative versus positive ocular carriers

CarrierCationicmolecule*

In vitro/in vivo Model Method Result References

Liposomes SA In vitro Cornealpermeability

Perfusion chambers Greatest corneal uptake ofcationic liposomes. Increasedcorneal permeability for drugsencapsulated in positiveliposomes

(Schaeffer andKrohn, 1982)

Decreased corneal permeabilityfor drugs encapsulated inpositive liposomes

(Law et al., 2000)

In vivo Awake rabbits Aqueous humor sampling Increased bioavailability of drugencapsulated in positiveliposomes

(Law et al., 2000)

Efficacy Improved efficacy of drugencapsulated in cationicliposomes

(Nagarsenker et al.,1999)

(El-Gazayerly andHikal, 1997)

(Meisner et al., 1989)

Oil-in-wateremulsion

SA In vitro Cornealwettability

Corneal contact anglemeasurement

Lower contact angle and betterspreading coefficient for thepositive emulsion

(Klang et al., 2000)

In vivo Awake rabbits Ocular tissue sampling Improved bioavailability ofcationic emulsion inanterior ocular tissues

(Abdulrazik et al.,2001)

Aqueous humor andocular tissue sampling

Increased posteriorbioavailability of drugencapsulated in cationicemulsion

(Klang et al., 2000)

Nanocapsules CS In vitro Cornealpermeability

Confocal microscopyPerfusion chambers

Retention of the cationiccolloid in the superficialcorneal layers. Increasedcorneal permeability formarker encapsulatedin positive nanocapsules

(De Campos et al.,2003)

CS and PLL In vivo Awake rabbits Aqueous humor andocular tissue sampling

Improved bioavailabilitywith CS coating but notwith PLL compared withthe negative colloidcounterpart

(Calvo et al., 1997)

The list is exhaustive only for cationic nanoparticles and emulsions (see references for exhaustive literature on cationic liposomes (Meisner and Mezer, 1995;Kaur et al., 2004)).* SA: stearylamine, CS: chitosan, PLL: poly-L-lysine.

L. RABINOVICH-GUILATT et al.626

effective than with the corresponding neutral preparation.

Moreover, adding a cationic charge to the vesicles was

comparable in its effect to increasing the formulation

viscosity (which is a common way of increasing drug

residence time to the eye surface) (Nagarsenker et al.,

1999). Also for acetazolamide, the reduction in intraocular

pressure following topical application to rabbits was

enhanced by the presence of a cationic charge on the

liposome surface (El-Gazayerly and Hikal, 1997).

Meisner et al. evaluated the activity of either a

hydrophilic or a hydrophobic form of atropine, encapsu-

lated in neutral, anionic and cationic liposomes (Meisner

et al., 1989). They found, as expected, that for both types of

molecules the pupil dilatatory effect was prolonged by

liposomal encapsulation in the order of positive . neutral

or negative . solution. When comparing the two forms of

atropine incorporated into the same SA-liposomes, the base

form displayed a more prolonged effect than the sulphate

salt, corroborating the hypothesis that independently of

the initial effect of the carrier, the final bioavailability

(and hence biological activity) depends on the drug

character. In the same study, similar elimination constants

from the anterior ocular tissues were observed for atropine

in solution and in liposomes, leading to the conclusion that

the increased efficacy was due to an augmented corneal

loading effect and not to a sustained release of the drug, and

that the drug was no longer associated with the liposomes

when it reached the anterior eye.

Emulsions

The contact angle between freshly excised corneal tissue

and a drop of positive emulsion was 50% lower than that

of negatively charged emulsions, together with a higher

spreading coefficient (Klang et al., 2000). These improved

physicochemical parameters could tell us more in details

about the physicochemical mechanisms leading to the

increased residence time of a cationic colloid. Surpris-

ingly, it was observed that the positive charge did not

modify significantly the in vivo anterior (cornea and

conjunctiva) indomethacin distribution in respect to the

drug solution or to the negative formulation. In contrast,

an improved relative drug bioavailability in both aqueous

humor and sclera-retina tissues was observed.

On the contrary, when anionic and cationic emulsions

similar in size and composition (except the charged lipid)

containing cyclosporine were administrated to rabbits, the

positively charged formulation produced significantly

higher drug levels at the ocular surface (cornea and

conjunctiva), and acted as a reservoir up to 8 h post-

administration (Abdulrazik et al., 2001). Elevated

posterior (sclera-choroid-retina) ocular drug levels were

also determined which were not correlated to higher

systemic concentrations. Increased transconjunctival

and scleral permeation was proposed by the authors to

explain the high optic nerve concentrations, as already

described for other drugs (Ahmed and Patton, 1985;

Chien et al., 1990).

Nanoparticles (NP)

The influence of the charge on the interaction of NP with the

ocular mucosa was investigated by De Campos et al., who

evaluated the effect of chitosan (CS) coating on the ex vivo

transcorneal flux and corneal uptake of poly-1-caprolactone

NC containing rhodamine (De Campos et al., 2003).

The single presence of CS-NCs in physical mixture with

rhodamine was enough to augment the transport of the free

dye, indicating a potential role of CS-NC in the modulation

of the epithelial tight junctions, i.e. penetration enhance-

ment. However, as the encapsulated rhodamine permeated

to a greater extent than the single physical mixture of empty

NC and free marker, the authors concluded that the colloidal

nature of the carrier also affected the corneal uptake. When

comparing positive (coated) with negative (uncoated) NC,

the former exhibited increased rhodamine transcorneal flux

at early times suggesting a faster corneal interaction.

However, when the corneal tissue uptake was assessed,

a plateau in the incorporated rhodamine amount was found,

indicating that behind a certain concentration all the negative

sites of the ocular mucosa responsible for the interaction

might be occupied by the positive moieties of the

polysaccharide and that the electrostatic attraction could

be a saturable mechanism.

Consistent in vivo results were obtained for the same

systems containing indomethacin, with almost doubled

corneal and aqueous humor drug concentrations for

the CS-coated vector compared to the uncoated one

(Calvo et al., 1997).

Although all the above cited studies demonstrate the

supremacy of cationic vectors over anionic or neutral

ones, no definitive conclusion can be drawn regarding the

advantage of one type of carrier over another.

PHYSICOCHEMICAL PARAMETERS TO TAKE

INTO CONSIDERATIONS WHEN DEVELOPINGAN OPHTHALMIC CATIONIC CARRIER

When developing a new ocular drug delivery system,

several choices have to be made (Table II). In this chapter

the most important criteria for the selection of the

formulation parameters are summarized.

Type of Carrier

Compared with other potential systems of controlled drug

delivery such as implants or inserts, colloidal carriers

present the advantage of easy administration in a liquid

form. There is evidence that the colloidal character of the

carrier improves corneal drug penetration independently of

its nature or charge (Calvo et al., 1996a,b; Muchtar et al.,

1997; Klang et al., 2000; De Campos et al., 2001).

The administration of active compounds in a colloid system

augments their ocular residence time compared to free

drug, as the nasolacrimal clearance of any particle is

decreased (Fitzgerald et al., 1987) and as particles augment

CATIONIC VECTORS IN OCULAR DRUG DELIVERY 627

interactions with the corneal surface. Of course, the choice

of the carrier will depend on the drug physicochemical

character (as described previously no substantial diversity

was found between the different vectors disposition) as a

good loading capacity of the active compound is

imperative, to allow reducing the instilled dose. Significant

improvement of the ocular bioavailability can be obtained

simply by reducing the instilled volume and consequently

the drainage rate, as demonstrated by Chrai et al. (1973).

This strategy results in both less waste of pharmaceutical

product and lower systemic undesired effects.

While liposomes were found convenient for the

formulation of both hydrophilic (Meisner et al., 1989;

Velpandian et al., 1999; Law et al., 2000) and lipophilic

molecules (Meisner et al., 1989; Meisner and Mezer,

1995) as well as NS (Pignatello et al., 2002a) and (De

Campos et al., 2001; Pignatello et al., 2002a), or NC

(Zimmer and Kreuter, 1995) and (Calvo et al., 1997; De

Campos et al., 2003), emulsions are specifically devoted

to the administration of lipophilic drugs (Muchtar et al.,

1997; Klang et al., 1999).

Fate of the Carrier Following Administration

Most of the studies performed with colloids have shown

very limited penetration of the intact carrier into the

corneal epithelium (Zimmer et al., 1991; Calvo et al.,

1994; Calvo et al., 1996a,b; De Campos et al., 2003) or

none (Singh et al., 1993). This is not surprising as the first

two cellular layers of the epithelium are keratinized and

impermeable to almost any compound. A complete

permeation of colloids through the cornea into the

aqueous humor as well as a penetration through

intercellular junctions was never observed, leading to

the conclusion that if a general uptake mechanism for

small particles does exist in precorneal tissues, there is no

transport into deeper layers of the epithelium.

Hints of further discrimination between free or

encapsulated drug permeation can be found by comparing

the drug aqueous humor kinetics. Increasing the contact

time allows for an extension of the time permitted for the

absorption process. As a result, the aqueous humor levels

continue to increase beyond those obtained for the simple

solution (Cmax augments) and tmax is shifted to a later time

as shown for cationic liposomes (Law et al., 2000) and

emulsions (Abdulrazik et al., 2001). The magnitude of the

tmax shift is determined by the degree to which drainage

loss is postponed. In contrast, the drug elimination rate

from the aqueous humor will be the same for both the

solution and the colloidal form since it concerns in both

cases the same free form of the drug (Meisner et al., 1989).

In case the colloid is absorbed intact, the same variations

in Cmax and tmax may be observed, as particle penetration

is a slow process. In this situation however, the

elimination rates will differ. Unfortunately, complete

pharmacokinetic profiles are hardly performed due to the

great investment that they represent.

Size of the Carrier

In contrast to the delivery of individual molecules, where

the passive diffusion through the cornea is expected to be

inversely proportional to their size (Hamalainen et al.,

1997), the corneal permeation of colloids follows other

mechanisms. The extent of corneal permeation as a

function of the colloidal size has not been systematically

investigated yet.

Two pathways are available for particles to diffuse

intact across the epithelium: the paracellular route (i.e.

between cells), which is a water-filled pathway impeded

by gap and tight junctions and is favored by hydrophilic

character; and the transcellular route (i.e. across cell

membranes) which involves passage across cell mem-

branes. As the narrow part of the corneal epithelial gap

junction is estimated to be only 0.8 nm (Edward and

Prausnitz, 2001), it is not surprising that in any of the

above cited studies, no particles were detected in the

intercellular junctions. Then, the transcellular route is

considered to be the only possible for intact colloids

translocation (Fig. 4).

It is widely recognized that colloids enter the cells

through endocytosis, even if liposomes may have putative

additional internalization way by direct phospholipid

integration and fusion with the cell membrane (da Cruz

et al., 2001). When dealing about cationic vectors, the

term “adsorptive endocytosis” can be borrowed from the

non-viral transfection domain, another field in which it is

generally accepted that a positively charged complex is

essential for cell binding prior to internalization (Almofti

et al., 2003).

Three types of endocytosis may be considered (Fig. 4).

The first one, phagocytosis, occurs only in specialized

cells such as macrophages and neutrophils and results in

TABLE II Criteria for the selection of optimal formulation parameterswhen developing an ophthalmic colloidal drug delivery system

Factor Preference

Drug Preferentially lipophilic (log K 2–3).*Non-ionizable lipophilic compounds willconcentrate into the corneal epithelium,while ionizable lipophilic ones will partitionateinto the aqueous humor

Vector type Depends on encapsulated molecule. Shouldallow a high loading dose to reduce theinstilled volume

Carrier size Lowest as possible to facilitate corneal uptakeand passage

Carrier charge CationicpKa of cationic

moleculeShould guarantee ionization in the post-

administration microenvironmentOsmotic pressure Isotonic with physiological fluids to avoid irritation

and lacrimation. Glycerol preferred as isotonicagent to avoid charge shielding by salts

pH Close to physiological pH to avoid irritation andlacrimation. If buffering is necessary, the lowestpossible buffer concentration is to be used(,0.1 M)

* K: octanol/buffer pH 7.4 partition coefficient.

L. RABINOVICH-GUILATT et al.628

the formation of phagosomes. Fluid-phase endocytosis or

pinocytosis is a receptor-independent process and takes

place continuously in almost all cells, involving the

internalization of the soluble material in a macropino-

some. Lastly, the receptor-mediated or clathrin-coated pits

endocytosis, in which the ligand binds specifically to its

receptor, concentrates in a clathrin-coated region at the

cell membrane and is internalized in receptosomes.

It should be noted that receptor-mediated endocytosis is

many thousand times more efficient than simple

pinocytosis in enabling the cell to acquire the macro-

molecules it needs. It is generally assumed that particles

up to 100–200 nm can be internalized by both pinocytosis

and receptor-mediated endocytosis, while larger particles

(.1mm) have to be taken up by phagocytosis.

Data concerning the general interaction of colloidal

carriers with the corneal epithelium is not rich; most of

them concerning anionic NP, and actually only few studies

performed by confocal scanning microscopy have shown

intact delivery systems in corneal epithelial cells. In vitro

and in vivo studies confirmed that after a 2 h contact with

the cornea, negatively charged poly-1-caprolactone NC of

250 nm could be detected at 5mm deep from the corneal

surface (Calvo et al., 1994). Confocal microscopy images

suggested an endocytotic mechanism, but it should be

noted that this infiltration extent remains limited to the

first cell layer of the epithelium.

Further studies have shown that the inner structure or

specific composition of the colloid had no effect on this

behavior, as similar penetration extent was observed for

200 nm NC, NP and nanoemulsions (Calvo et al.,

1996a,b). In the only published study assessing the charge

effect on the ex vivo corneal cell internalization, CS-

coated NC were retained at a more superficial level than

the uncoated ones (De Campos et al., 2003). While the

authors suggested that improved electrostatic interaction

with the epithelium was responsible for this reduced

permeation, the fact that the cationic NC (465 nm) were

twice as big as the negatives ones (246 nm) could be

relevant too.

Trying to elucidate the endocytotic mechanisms,

Qaddoumi et al. have shown that 100 nm poly

(DL-lactide-co-glycolide) (PLGA) NP were internalized

in primary cultured rabbit conjunctival epithelial cells

independently of clathrin and caveolin-1-mediated path-

ways (Qaddoumi et al., 2003) and in the same cells,

300 nm CS-NP uptake was significantly reduced under

conditions that blocked active transport processes

(Diebold et al., 2003). In other epithelial cell lines, the

endocytosis of CS-NP was preceded by non-specific

interaction of the ligand with the cell membrane, not

necessarily electrostatic but also hydrophobic (Behrens

et al., 2002; Huang et al., 2002).

Choice of the Cationic Molecule which confersthe Charge

Most studies have used stearylamine (SA) to impart a

positive charge to liposomes or emulsions, while cationic

polymers such as CS have been incorporated into NP or

NC. Recently, the physicochemical characteristics of new

cationic emulsions containing oleylamine (OA) with

potential in ophthalmic delivery were examined too

(Rabinovich-Guilatt et al., 2004).

Evidence that the specific nature of the cationic molecule

may be responsible for improved uptake properties was

supplied by Calvo et al. who showed that two different

types of cationic indomethacin loaded NC (coated with

poly-L-lysine or CS correspondingly) resulted in comple-

tely different drug kinetics profiles (Calvo et al., 1997),

possibly due to the added CS mucoadhesive properties

(Lehr et al., 1992).

The effective cationic charge density at the vector

surface is determined by its surface concentration, its pKa

and the surrounding microenvironment. It is worth selec-

ting the pKa of the molecule rendering the positive charge

to the vector in such a way to obtain the required ionization

degree at the ocular pH. In general, the positive character of

the carrier is confirmed by measuring its zeta potential, but

this is often performed after dilution in media that are far

FIGURE 4 Cell endocytosis pathways of colloids into the corneal epithelium.

CATIONIC VECTORS IN OCULAR DRUG DELIVERY 629

from the eye environment regarding pH and ionic strength.

A colloid which shows highly positive zeta potential when

measured in distilled water or 10 mM NaCl will exhibit

much lower values in the presence of salt, and it may even

reverse its charge at physiological pH.

Another factor influencing the cation selection is

the toxicity. For cationic lipids, Taniguchi et al. demon-

strated that following nine instillations every 15 min,

SA-containing vesicles (0.5 mg/ml in the final formulation)

did not produce more ocular irritation than

neutral liposomes as evaluated by the Draize test and

corneal histological examination. However, in a

more sensitive test the positively charged preparation

increased the blinking count in the rabbit, suggesting

that it may cause pain or discomfort (Taniguchi et al.,

1988). Shaeffer and Krohn have demonstrated that

the increased penicillin corneal flux observed with

SA-liposomes (1.15 mg/ml in final formulation) was not a

result of corneal epithelial cell damage (Schaeffer and

Krohn, 1982). Similarly, in a subchronic toxicity study

performed in rabbits, a 3 mg/ml SA-emulsion was found to

be safe for ocular topical administration (Klang et al.,

1994). Tolerance studies in rabbits eyes eight times per day

for 28 days of a 1 mg/ml OA ophthalmic emulsion have

shown that the product is well tolerated, a finding which

was further confirmed in a clinical trial in healthy

volunteers.

Regarding cationic polymers, up to 15 mg/ml of CS

(in solution and colloidal form) were well tolerated in a

rabbit subchronic study, as demonstrated by an ocular

irritation test, confocal laser scanning ophthalmoscopy

combined with corneal fluorescein staining and histo-

logical analysis (Calvo et al., 1997; Felt et al., 1999). The

same conclusion could be drawn from another study

concerning NP coated with Eudragitw RS100 and RL100

(copolymers of poly(ethylacrylate, methyl-methacrylate

and chlorotrimethyl-ammonioethyl methacrylate)

containing quaternary ammonium groups), which showed

no particular sign of toxicity or irritation following 12

consecutive instillations as assessed by the Draize test and

slit lamp examination (Pignatello et al., 2002a,b).

Finally, the stability of the cation to the manufacturing

procedure, especially to the sterilization process

should be considered. Chemical and gamma sterilization

are reported to degrade CS in solution, while filtration

of the final formulation might be difficult due to its

viscosity.

External Dispersing Phase

pH and Ionic Force

General Considerations Concerning Ophthalmic

Products

Concerns about the solution media of any ophthalmic

product are restricted in most cases merely to the

adjustment of ionic force and pH to physiological values.

With normal tear osmotic pressure of 0.9% NaCl and pH

of 7.4, the eye can tolerate osmotic pressure and pH ranges

of 0.6–1.3% NaCl and pH 6.0–9.0, respectively. In this

view, it is noteworthy that an irritating formulation will

not only reduce the patient’s compliance, but it will also

provoke faster ocular clearance by stimulating tears

production. Indeed, Ahmed and Patton demonstrated that

the first order constant associated with the corneal

absorption was inversely proportional to the fluid volume

in the donor compartment, i.e. larger lacrimal fluid volume

in the precorneal area will reduce the absorption rate

(Ahmed and Patton, 1984).

Specific Considerations Concerning Cationic

Carriers

When administering a cationic carrier, the impact of the

dispersing media is a critical point, as the degree of

ionization of the instilled vector and hence its interaction

with the cornea, will be determined by the lacrimal fluid

pH, which is itself significantly influenced by the instilled

solution. While for individual molecules the effect of the

formulation pH on the drug penetration was extensively

investigated (Schoenwald and Huang, 1983; Ahmed and

Patton, 1984; Small et al., 1997), the influence on the

absorption and/or efficacy of cationic vectors was never

investigated.

Regarding the effect of the formulation on the lacrimal

pH, many researchers have tracked the pH evolution up to

1 h post-instillation (Longwell et al., 1976), but only the

first few minutes represent the critical period for drug

uptake by the cornea. Independently of the initial pH shift

extent, the restoring to physiological values is fast (the

greater the pH alteration is, the faster the induced lacrimal

rinse) (Patton and Robinson, 1976). Phosphate buffer

strengths of 0.07–0.1 M at pH 4.5 were found efficient

enough to acidify the tears for 15 min. Administration of

50ml of HCl pH 4 reduced immediately the tear film pH

by 0.5 units (Longwell et al., 1976), while 25ml of 67 mM

phosphate buffer pH 4.5 causes a drop of 1.6 units (Ahmed

and Patton, 1984).

Consequently, if a non-physiological pH environment is

required in the cornea for stability, absorption and/or

ionization reasons and if buffering is indispensable,

the minimal buffering capacity needed should be

employed. Excessive buffer strengths will indeed

cause lacrimation, without improving the absorption.

In addition, high ionic strength can have other effects

as demonstrated by Rojanasakul and Robinson who

have observed that increasing the ionic strength of

the immersion solution from 10 to 160 mM reduced the

in vitro corneal selectivity to cations comparatively to

anions, probably by amplifying the shielding of the

charges responsible for the electrostatic attraction

(Rojanasakul and Robinson, 1989).

Viscosity

In order to slow further the clearance from the eye,

a number of viscosifying agents such as celluloses,

L. RABINOVICH-GUILATT et al.630

polyvinyl alcohol or polyacrylic acid might be added to

the formulation. It should be noted though that even when

the precorneal residence time might be augmented it is

only for the first second post-administration (Zaki et al.,

1986), and their clinical effect was demonstrated to be

very limited (Ding, 1998).

Also the ability of CS as a potential viscosifying

excipient was evaluated, resulting in a 3–5-fold improve-

ment on the mean precorneal residence time of a solution

of tobramycin. However, as the increase in residence time

was not correlated to the viscosity values it can be

concluded that other physicochemical properties of the

excipient may be more important than considerations of

viscosity (Felt et al., 1999).

Encapsulated Drug

The corneal permeability of any molecule will

potentially be improved following incorporation into

a particulate system, as the precorneal residence

time is prolonged. An additional general advantage to

use colloidal systems over solutions of the same

drug content is the fact that the corneal epithelium is

confronted to a more concentrated internal dispersed

phase, resulting in an augmented gradient driving force,

according to Fick’s law.

If the drug dissociates from the carrier before crossing

the cornea as it was previously discussed, its intrinsic

permeability will shape its further penetration. For

instance, administration of 25 mg of indomethacin

encapsulated in CS-coated NC resulted in an estimated

aqueous humor concentration of 100 ng/ml (Calvo et al.,

1997), while for a similar dose of cyclosporine (16mg)

encapsulated in a related system, less than 10 ng/ml were

obtained in the aqueous humor (De Campos et al., 2001).

The physicochemical property that probably has the

most important influence on corneal penetration of a drug

is its lipid and water solubility. While water solubility is

needed to assure both satisfactory precorneal concen-

tration and stroma permeability, lipophilicity is required to

epithelial corneal penetration (Maren and Jankowska,

1985). Consequently, a lipophilic non-ionizable com-

pound will likely accumulate into a depot in the corneal

epithelium following topical administration without going

across the stroma (Maren and Jankowska, 1985), while an

ionizable lipophilic drug would have more chances to

traverse through consecutive partitioning equilibriums

into the aqueous humor (Conroy and Maren, 1995).

Thus, in vitro permeability studies in rabbit

cornea have found that the optimum apparent

partition coefficient in octanol/buffer (pH 7.4) for corneal

penetration of drugs is in the range of 100–1000

(Schoenwald and Ward, 1978; Huang et al., 1983;

Schoenwald and Huang, 1983). Moreover, higher permea-

bility coefficients were associated with shorter lag times

of permeation which could avoid the rapid drainage

(Suhonen et al., 1991) and non-productive absorption

(Chien et al., 1990; Hamalainen et al., 1997).

OTHER BIOLOGICAL CONSIDERATIONS

Disease to Treat

While the target for the treatment of surface eye diseases

such as infections (conjunctivitis, blepharitis, keratitis

sicca, etc) is the surface of the ocular mucosa, it will be the

ciliary body and/or deeper tissues in the case of intraocular

diseases such as glaucoma or uveitis. Consequently,

depending on the treated disease, the full passage of the

active entity up to the anterior chamber is needed or just

corneal uptake will be required, feature for which a

specific vector might show a particular affinity (Schaeffer

and Krohn, 1982).

There is evidence that inflamed eye tissues display

increased permeability to both colloidal particles and

released molecules, most likely by altering ocular protein

and fluid contents or due to physical breakdown of the

epithelium, emphasizing the need for both pharmacoki-

netics and efficacy studies to be performed in experimen-

tal animals disease models. Diepold et al. have found for

instance 3–5 higher NP concentration in inflamed ocular

tissues than in normal ones. This alteration could be

attributed to increased precorneal protein binding, a

potential partial blockade of the nasolacrimal duct or a

higher tissue hydration (Diepold et al., 1989).

In Vivo and In Vitro Models

It is difficult to avoid artifacts in both in vitro and in vivo

research in ophthalmology. In vitro studies neglect main

factors as drainage and non-productive absorption which in

physiological conditions account for the main elimination

pathways. Ex vivo corneal permeability studies in which the

vector is in contact with the cornea for 2 or 4 h do not reflect

the physiological condition either, and should be used only

to better understand in vivo results.

Most in vivo studies are done as single dose regime with

obvious suboptimal conditions regarding corneal and ocular

concentration steady states. Many animal experiments are

still performed in anesthetized animals, where tear

production is reduced (Chrai et al., 1973; Patton and

Robinson, 1976). In clinical studies conversely, the collec-

tion method of the tears (roughly the only possible sampling

physiological fluid) may affect their production and

composition (Baeyens and Gurny, 1997; Pandit et al., 1999).

An essential parameter which should be investigated in

ocular pharmacokinetic studies is the drug peripheral

concentration, as an increased systemic absorption will

lead to higher ocular tissue levels, especially in the

posterior eye, and to pharmacological effects in the

untreated eye when unilateral application is employed.

CONCLUSION

For an ophthalmic dosing system to provide optimum

ocular drug penetration, a balance must be achieved

CATIONIC VECTORS IN OCULAR DRUG DELIVERY 631

between the requirement of both the drug and its vehicle.

The only suitable means to attain this goal is to determine

the mechanism(s) of vehicle effects and to relate them

quantitatively to the mechanism of ocular penetration of

the drug. In this view, a rational approach of the

formulation must take into consideration numerous

physicochemical as well as biological parameters to

improve drug delivery to the eyes.

Acknowledgements

We thank the Association Nationale de la Recherche

Technique (ANRT) for supporting Laura Rabinovich-

Guilatt with a CIFRE convention.

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